Flow analysis has proven to be an important technology for the analysis of discrete targets. The applications of this technology include cellular assay to investigate a variety of cellular features including DNA content, specific nucleic acid sequences, chromatic structure, RNA content, specific antigens, surface receptors, cell morphology, DNA degredation and other assay targets. The targets of a flow cytometer may be multicellular organisms (e.g. microfilaria), cellular aggregates, viable cells, dead cells, cell fragments, organelles, large molecules (e.g. DNA), particles such as beads, viral particles or other discrete targets of this size range. The term “cells”, as used throughout, is used to refer to such discrete targets. This technology has a number of different applications, including diagnostic, clinical and research applications.
Flow cytometry measures targets flowing through an analytical region in a flow cell. In the flow cell a core stream is injected into the center of a sheath flow stream flowing at a constant flow rate. The core stream is a liquid sample, which may be injected from a sample tube. Injection generally requires insertion of an aspiration tube into the sample tube and pressurization of the head above the liquid in the sample tube such that sample liquid is pressure driven from the sample tube into the injection tube.
The flow stream is directed into a tapered portion of the flow cell body and through an analytical region. In one design, the stream is directed through a nozzle and analyzed in air. In a second design, the stream is directed through a channel for analysis.
Analysis takes place by optical interrogation of particles as each particle passes a detection region. In most systems, one or more laser beams are directed by steering mirrors and illumination lenses through the analytical region. If more than one laser are used, a dichroic stack may be used to combine the beams and direct the beams through the stream to be analyzed.
Some of the light passing through the analytical region will be scattered by particles. Detectors measure the intensity of forward and side scatter. In addition, the illumination beam will excite fluorescence from target particles in the flow stream that have been labeled with a fluorescent dye. Emitted fluorescence is collected by a collection lens and transmitted to detection optics. The detection optics separate the collected light (e.g. using filters and dichroic mirrors) into light at specific wavelengths. Light at specific wavelengths, or within specific wavelength ranges, are detected by individual light detection devices (e.g. photomultiplier tubes). The signal from the various detectors is sent to a data processor and memory to record and characterize detection events.
In addition to analysis of particles, flow cytometer systems may also be designed to sort particles. After leaving the optical analysis region, the flow stream may be separated into droplets. One common method of droplet generation is to vibrate the nozzle from which the flow stream emerges. This may be done by vibration of the nozzle alone, or vibration of the entire flow cell. The resultant separated droplets adopt a spacing which is a function of the stream velocity and the vibration wavelength. Droplets containing the target of interest are charged by a charging device such as a charging collar. The charged droplets are directed between two charged deflection plates, which angularly deflect charged droplets. The deflected droplets are then collected in containers positioned in the path of falling deflected particles.
Known flow cytometry similar to the type described above are described, for example in U.S. Pat. Nos. 3,960,449; 4,347,935; 4,667,830; 5,464,581; 5,483,469; 5,602,039; 5,643,796 and 5,700,692. All references noted are hereby expressly incorporated by reference. Commercial flow cytometer products include FACSort™, FACSVantage™, FACSCount™, FACScan™, and FACSCalibur™ systems all manufactured by BD Biosciences, the assignee of the instant invention.
The described system presents a number of advantages for the analysis of particles (e.g. cells), allowing rapid analysis and sorting. However a number of limitations to the system exist.
Alignment
The system requires precise alignment of various elements to function properly. The lasers must be precisely positioned to properly direct light to the objective. To aid in this positioning, the laser or other illumination source is commonly mounted on an x-y-z stage, allowing three-dimensional positioning of the laser. The steering mirrors for the laser beams must be precisely positioned to properly direct the illumination beam to the objective. This generally requires that the mirrors be mounted to allow for angular adjustment. The illumination lens system must be exactly positioned such that the illumination lens focuses the illumination light onto the target area. This lens is also generally mounted such that it can be repositioned along the x-y-z axes.
The flow cell must be positioned such that the angle at which the illumination beam impinges the flow stream and the distance from the flow stream to the illumination lens does not change. Commonly the flow cell is mounted on a stage, which allows x-y-z positioning of the flow cell. In addition the stage holding the flow cell may also allow for angular repositioning of the flow cell (e.g. α and θ positioning). This angular adjustment is critical for sorting, which requires precise prediction of the sort stream direction. In addition, the optics used for detection of scattered light and fluorescence also must be properly aligned.
The stream in air jet must also be aligned, to ensure that the stream in air is directed in the intended direction. This alignment is effected by angular rotation of the flow cell. This alignment is additionally important if the optical interrogation of the stream takes place in a stream-in-air. The alignment procedure for a stream in air system requires first locating the stream-in-air with respect to both the illumination and the light collection optics and then focusing each of these components on a location within the stream in air.
Alignment requires user time and considerable user expertise. At times it is difficult to determine which element requires adjustment. Set up of the instrument generally requires a diagnostic of alignment with elements realigned by repositioning as needed. This occurs at least once a day, more frequently if an element is replaced or removed. Realignment necessitates both instrument down time and user time and expertise. The time required to perform the alignment procedure is highly dependent on both the condition of the system and the skill of the operator. In addition, the need for constant realignment reduces the repeatability of system performance.
A few attempts have been made to address the problem of the need for repeated alignment of some elements of a flow system. U.S. Pat. Nos. 5,973,842 and 6,042,249 to Spangenberg disclose an optical illumination assembly for use with an analytical instrument. This assembly may include an illumination source (e.g. a laser), a spatial filter, a beam shaping aperture and a focus lens. All elements are illumination optical elements, not the flow cell or light collection elements. Each component is mounted on a plate, frame or mounting cylinder, which in turn are mounted on a platform. Each of the plates or frames is movable along two axes by micrometer adjustments using adjusters with opposing spring plungers. Following an initial adjustment, the plates or frames are secured into a fixed location using screws or other devices to fix the plates or frames into place. The adjusters or springs are removed once the frames or plates are secured. The focus lens would be mounted such that it would be moved along 3 axes (x-y-z movement) and subsequently also be fixed into a location. This allows fixation of the light generation and illumination optics. However, the cuvette would still be adjusted to be positioned at the focal spot of the illumination. This would be required on a routine basis.
U.S. Pat. No. 4,660,971 discloses an illumination configuration in which a focus lens is in contact with a flow cell. A spring biases the lens against a housing, positioning the lens at a selected focal length from the flow cell. This maintains a relative axial position between the lens and the flow cell.
These references, while providing a method in which some of the issues relating to the alignment of the illumination optics are addressed, do not provide a method in which the flow cell and the light collection optics may also be fixed. Fixing all of these elements significantly further simplifies the alignment of the instrument.
Illumination Power
A number of different features in a common flow cytometer setup result in loss of illumination intensity or loss of intensity of collected light. To compensate for these losses generally requires increased illumination power. This requirement for increased power requires expensive and bulky liquid-cooled lasers that provide sufficient power to overcome losses and still allow sensitive target detection. These sources of loss include:    1. Optical interrogation using a stream-in-air. The gross cylindrical geometry of a stream of liquid in air acts as a lens both reflecting and refracting illumination light. This high index of refraction is more pronounced in smaller diameter streams. This refraction makes illumination less efficient and distorts light scatter. To mitigate this effect of scatter distortion an obscuration bar is positioned between the stream in air and the light scatter detector. In some systems, this rectangular obscuration bar may be rotated to block additional amounts of light scatter across a greater area, blocking additional light from narrow angles from reaching the scattered light detectors.    2. Use of dichroic mirrors to combine illumination beams. Each dichroic mirror is not able to perfectly reflect or transmit a light beam. As the beam is reflected or transmitted some light is lost. This loss ranges from 10–20% of beam power (5–10% if beam is reflected; 10–15% loss for transmission through a dichroic mirror), more if the dichroic is not perfectly aligned. A laser beam that is reflected by a steering mirror through two dichroic mirrors to combine three beams could lose 40% or more of the laser's power.    3. Losses in collection of fluorescence. The amount of collected fluorescence can be limited by the optical properties of the flow cell and the collection lens. The geometry of the flow channel and the flow cell define the numerical aperture from which the system is able to collect light. The transition from the flow cell to the collection lens could allow refraction of light and loss of signal as the emitted fluorescence travels through the flow cell, into air, and then into the collection lens. The high index of refraction during material transition results in the loss of collected light. In some systems this loss is mitigated by physical coupling of the flow cell to the collection lens. However, this coupling would be greatly simplified if the flow cell were in a fixed location.Droplet Generation
Droplet generation has required vibration of some part of the flow cell, generally either the nozzle or the entire flow cell. Vibration of the entire flow cell can result in alignment difficulty as well as additional light scatter created by the vibration. In addition, if the optical analysis is performed in a stream-in-air, the drop-drive perturbations cause undulations on the free surface of the stream. This causes a constant alteration of the light paths into and out of the jet of liquid, making measurement of scatter and focusing of the illumination beam more difficult.
U.S. Pat. No. 6,133,044 provides one alternative to the vibration method of droplet generation. This reference describes a device in which an oscillator is included within the nozzle volume or otherwise is undirectionally coupled to the sheath fluid. The tapering of the nozzle amplifies the oscillations, which are transmitted as pressure waves through the nozzle volume to the nozzle exit. This results in the formation of droplets. The nozzle is directionally isolated to avoid vibration of the entire flow cell or nozzle and limit the oscillations to forming pressure waves in the flow stream.
Optics Positioning Limitations
Ideally, the flow cell would be materially joined to the light collection optics to prevent the loss of collected light. One of the greatest losses of collected light occurs due to the transition between different materials that each have a different idex of refraction of light. The light refraction between different materials (e.g. air and glass) may be significant and the resultant light refraction makes the collection and measurement of scattered or fluorescent light difficult. This is mitigated by joining the flow cell to the light collection lens. However for the flow cell and the light collection lens to be coupled by a physical material would require that the two elements remain in a fixed location.
In addition, the need to guard the flow cell from damage (e.g. scratching of surfaces through which light passes) presents another motivation for keeping the flow cell at a fixed location.
Flow Cell Positioning Limitations
Sorting flow cytometers generate a stream of droplets in air and subsequently sort droplets containing target particles. The droplet stream is generated from a flow nozzle positioned at one end of a flow channel. A large degree of uncertainty in the nature of the stream of droplets is a common result of the way in which the nozzle is located to the flow channel. Most flow designs rely on the “self-aligning” tendency of a female conical structure at the nozzle inlet, which mates with an edge on a cylindrical structure at the flowcell outlet (i.e. the outlet of the flow channel). Typically an o-ring makes a seal between the nozzle conical structure and the flow channel cylindrical structure.
However, there are a few problems inherent with this approach. First, the o-ring has a compliance that aggravates the axial and angular tolerance stack-up associated with locating a conical surface about a circular arc. Second, the angular location of the nozzle about the axis of the flow cell is arbitrary. Third, the angular location of the o-ring about the axis of the flow cell is arbitrary. The first noted problem makes it difficult for a user to duplicate the mounting of the nozzle to a previous mounting configuration. The second and third noted problems make it impossible. Because the angular location of the nozzle and the o-ring are arbitrary, the nozzle is not formally constrained with respect to the flow channel (or the cuvette) through which the flow cell extends.
U.S. Pat. Nos. 6,263,745 and 6,357,307 to Buchanan et al. disclose a nozzle for sorting flat samples. This nozzle seats in a cylindrical recess in the flow cell. U.S. Pat. No. 6,133,044 discloses a removable nozzle for use with a flow cytometer. The nozzle seats in a cylindrical recess in the flow body and is held against a lip. An annular nut secures the nozzle to the body of the tapering flow cell. An o-ring positioned between the nut, the nozzle and the tapering flow cell provides a means for ensuring the axial orientation of the nozzle.
Cell Sorting
Cell sorting requires precise coordination of event detection, droplet generation and droplet tagging. If these procedures become even slightly out of coordination, the incorrect droplets could be charged for sorting or the system could fail to collect the desired particles or cells. For stream-in-air analysis and sorting, this process is simplified because the droplet stream is optically analyzed, droplets are generated and droplets charged all in a stream in air. However, as noted earlier, the stream-in-air sorting produces a decreased signal from cells or particles sorted and the circular stream of liquid can cause both illumination light and scattered light to be reflected or refracted.
Sorting using a system in which analysis is done in a channel also presents challenges. When the liquid moves from an analysis channel and subsequently through the nozzle the velocity of the particles changes, as the liquid flow accelerates at the narrow nozzle. The coordination of flow must account for this change in flow rate.
U.S. Pat. No. 6,372,506 to Norton discloses an apparatus and method for determining drop delay. Drop delay is the time that elapses between detection of a target at an analytical region to the time at which a sorting condition (e.g. a charging potential) is applied to the droplet. As the droplets are formed they are analyzed to determine whether the drop delay is correct. The droplets are analyzed to determine if the target detected at an analytical region is within the droplet to which the sorting condition is applied.
As fluid enters a channel, flow over a short distance can be modeled as “slug flow”, all liquid moves as a single front. This would be the case at the entrance of the neckdown region of the flow cell. As liquid moves along the length of the channel, the viscosity of the liquid produces a parabolic velocity profile. The velocity of the liquid flowing through the cuvette channel tube is fastest along the longitudinal axis of the tube. At the walls of the tube the fluid has no velocity. At any intermediate point between the walls and the center of the channel, the velocity of the fluid varies parabolically. This laminar flow results in a spreading of the distance between particles at different distances from the tube center as the particles move through the stream. Particles in the exact center of the stream will move faster than the particles closer to the edges of the stream.
The laminar flow produces a spread of particles as the particles move through the channel. This can make sorting particles optically analyzed in a cuvette channel more difficult. If the velocity of a particle changes as the particle moves through the channel and optical interrogation occurs in the channel, the velocity of the particle at the point of optical interrogation and the velocity of the particle at the point of exiting the channel through a nozzle will be significantly different. Since prediction of the position of the particle depends on knowing the velocity of the particle, sorting particles becomes much more difficult if the velocity of the particle changes.
It is an object of the invention to provide a flow cytometer that requires alignment less frequently, and most preferably only at an initial instrument setup.
It is a further object that this system allows for efficient illumination and collection of light.
It is a further object that the losses of illumination light be reduced to allow for lower power lasers to be used for illumination.
It is a further object to provide a system that is easier to use and provides robust system performance.